RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

Abstract

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells

“…It has been known that the level of expression of transgene from a mammalian expression system correlates mainly with the expression vector, so that the use of an optimal expression vector is a primary requirement for high-level expression of transgene in mammalian cells. Expression vector is usually controlled by regulatory elements [18][19][20], which usually exists in non-coding regions, such as upstream enhancers and promoters and polyadenylation elements and terminator in downstream regions of the genes [21][22][23][24]. Introns, as non-coding DNA sequences, have the function to regulation gene expression in eukaryotes by a variety of mechanisms, such as enhancing RNA polymerase II process activity, promoting the interaction between splicing proteins and certain transcription factors, connecting and facilitating multiple types of RNA processing mechanisms and affecting nuclear mRNA export, translational efficiency and RNA decay [25][26][27].…”

Effect of promoter, promoter mutation and enhancer on transgene expression mediated by episomal vectors in transfected HEK293, Chang liver and primary cells

“…To further enhance the functions of promoters, various enhancer elements have been added upstream of the promoters [39]. The enhancers are cis-acting elements that can increase transcription levels.…”

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

“…hCMV‐MIE is prone to transgene silencing, which is associated with DNA methylation . Moreover, recombinant CHO cell lines with EF1α exhibited loss of productivity during long‐term culture . Therefore, to monitor the effects of Dnmt3a‐deficient CHO cells on transgene expression, we used 2 promoters, namely CMV and EF1α, to drive the recombinant gene expression.…”

“…The pWTY‐CMV and pWTY‐EF1α vectors, which contained either the CMV (589 bp) or the EF1α (1335 bp) promoters, were constructed by cloning eGFP as a report gene from pEGFP‐C1 (Clontech, USA) into the pIRES‐neo vector (Clontech) . The sequences of the elements were synthesized and sequenced by General Biosystems (Chuzhou, Anhui, China).…”